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INVESTIGACIÓN ORIGINAL
Inhibition of Helicobacter pylori growth by an Asteraceae family plant methanol extract.
Inhibición del crecimiento de Helicobacter pylori por un extracto de metanol de una planta de la familia Asteraceae.
David Espinosa-Ramos1, Ricardo Gomez-Flores1, Patricia Tamez-Guerra1, Ramiro Quintanilla-Licea1, Myriam de la Garza-Ramos2.
1
Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo
León. Ave. Universidad s/n. San Nicolás de los Garza, N. L. México.
66450.
2 Facultad de Odontología / Centro de Investigación y Desarrollo
en Ciencias de la Salud, Universidad Autónoma de Nuevo León. Calle
Dr. Carlos Canseco y Ave. Gonzalitos s/n, Colonia Mitras Centro, Monterrey,
Nuevo León, 64460, México.
ABSTACT
Helicobacter pylori is a spiral Gram-negative bacterium
associated with inflammation of the gastric mucosa, peptic ulcer, and gastric
adenocarcinoma; it is one of the leading causes of cancer death worldwide. Treatment
prescribed to patients with gastric ulcer has failed in many cases mainly due
to antibiotic resistance and important side effects such as taste disturbances,
vaginal candidiasis, and pseudomembranous colitis. Interest in botanical medicine
is increasing as a viable alternative to the traditional one. Plant leaves of
the Asteraceae family are used to treat diarrhea, ulcers and rheumatism. The
aim of this study was to evaluate the activity of aqueous and methanolic extracts
from plants of the Asteraceae family against H. pylori growth in vitro,
using the colorimetric tetrazolium bromide (MTT) reduction assay. We observed
that methanolic extracts from plants of the Asteraceae family showed up to 82%
H. pylori growth inhibition (MIC at 500 µg/mL).The results of the
present study contribute to the body of knowledge of medicinal plants with antimicrobial
potential, particularly against H. pylori.
Keywords: Helicobacter pylori •
Asteraceae family plant • Methanolic extracts • Aqueous
extracts.
INTRODUCCIÓN
H. pylori infects and colonizes the human stomach in
50% of the world’s population (Hongying et al., 2014). Chronic infection
in the human stomach is characterized by chronic inflammation. The development
of gastric adenocarcinoma, particularly of the intestinal type, is preceded
by the development of chronic gastritis, atrophic gastritis, intestinal metaplasia,
and dysplasia. In developing countries, 70 to 90% of the population becomes
infected before 10 years of age and more than 80% of adults and 50% of children
are colonized by H. pylori compared with 30% of adults and 10% of children in
developed countries (Dunn et al., 1997, Dye et al., 2002). Researchers have
mentioned the oral cavity as the main reservoir of intragastric H. pylori
and it is likely that oral health is directly related to infection or reinfection
with H. pylori (Cover et al., 1995). H. pylori has been detected
in saliva, plaque and periodontal disease patients by PCR, culture, urease test;
this organism can promote the development of lesions of the oral mucosa, particularly
recurring ulcer disease and lesions of the oral mucosa (Rlgglo and Lennon, 1999;
Nguyen et al., 1993). In México, seroprevalence of H. pylori is
higher than 50% (Alvarado-Esquivel et al., 2013). The first-line treatment option
for H. pylori consists of a 7 to 10 days regimen with protonpump inhibitor
(PPI), plus amoxicillin, and clarithromycin (Alahdab et al., 2014).The use of
clarithromycin increases resistance to H. pylori (Wu et al., 2014). Treatment
prescribed to patients with gastric ulcer fail in many cases mainly due to antibiotic
resistance, in addition to induced side effects. Interest in botanical medicine
has increased in recent years, physicians and people seem to show preference
for products that contain “natural extracts” instead of products based
on “synthetic” substances (Borchers et al., 2000, Torrado-Truiti et
al., 2003) The first report of plants against H. pylori was made in 1991
(Cassel-Beraud et al.,1991). There are reports of methanol and aqueous extracts
of Mexican plants that have inhibitory effect against H. pylori (Castillo-Juárez
et al., 2009).The plant compounds including polyphenols, flavonoids, quinones,
coumarins, terpenoids and alkaloids.The anti-H. pylori action mechanism,
including inhibition of enzymatic(urease, DNA gyrase, dihydrofolate reductase,
N-acetyltransferase, and myeloperoxidase) and anti-adhesion activities, high
redox potential and hydrophilic/hydrophobic natures of compounds (Wang, 2014).
The aim of this study was to evaluate aqueous and methanol plants extracts using
the MTT tetrazolium reduction assay as a viability test to assess direct effects
of these plant extracts against H. pylori growth in vitro.
MATERIAL Y MÉTODOS
Five grams of each sample were used
to prepare the extracts. For aqueous extracts, the plants were boiled for 10
min with 80 mL of purified water filtered and then lyophilized in freeze dry
system (LABCONCO Corp., Kansas City, MI) for about four hours. Once the sample
was obtained the amount of extracts was calculated. In the case of methanol
extracts the material was extracted for 72 hours in 80 mL of methanol. After
filtration the extracts were evaporated in a Speed Vac (Milford, MA). The plants
screened in the present study were Persea americana Mill (Lauraceae),
Pachycereus marginatus (DC.) Britton & Rose, a plant of the Asteraceae
family (its use is in the process of patenting), Ibervillea sonorae Green,
and Phoenix dactylifera Linn.
Bacterial strain and culture conditions
H. pylori standard strain ATCC 43504 was grown on Brucella
broth for a day at 37°C. The strain was identified by Gram staining morphology
and biochemical positive tests for catalase and urease.
Minimum inhibitory concentration
(MIC) determinations
The aqueous and methanol extracts were tested by MTT tetrazolium reduction assay
in 96-flat well microplates. The extracts were dissolved in Brucella broth to
obtain a final concentration 7.8, 15.6, 31.2, 62.5,125, 250, and 500 µg/mL.
A volume of 50 µL of H. pylori (2.5x105 bacteria/mL) was placed
in the plate wells and incubated. The MIC was determined using the MTT reduction
assay, being the lowest concentration of the extracts in the plate with no bacterial
growth. Before extracts were incubated, we added 15 µL of MTT and incubated
for 15 min, then 80 µL of DMSO were added to dissolve the formazan crystals.
The absorbances were measured in a microplate reader at 570 nm (DTX 800/880
Multimode Detectors, Fullerton, CA). All the experiments were performed in triplicate
and repeated at least three times. Tetracyclin was used as a positive control.
RESULTS
Methanolic and aqueous extracts of 5 different plants were tested in vitro for
their anti H. pylori activity. No aqueous extract of the 5 plants showed
in vitro effect against H.pylori ATCC 43504. Only methanolic extracts
from plants of the Asteraceae family showed inhibitory effect against H.
pylori. Such results are shown in Figure 1. The vehicle control did not
affect H. pylori viability.
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Figure
1. Inhibitory effect of methanol plant leaves extract of a plant of the
Asteraceae family.
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As
seen in Figure 1, the Asteraceae plant methanolic extracts showed up to 82.3%
growth inhibition of H. pylori ATCC 43504 at a concentration of 500 ìg/mL
(p<0.05), and the growth inhibition activity was in a concentrationdependent
manner.
DISCUSSION
The beneficial medicinal effects of plant materials typically result from the
combinations of secondary products present in the plant. In plants, these compounds
are mostly secondary metabolites such as alkaloids, steroids, tannins, and phenol
compounds, which are synthesized and deposited in specific parts or in all parts
of the plant (Parekh et al., 2005).
Although our aqueous extracts of Persea americana Mill (Lauraceae), Pachycereus
marginatus (DC.) Britton & Rose, a plant of the Asteraceae family, Ibervillea
sonorae Green, and Phoenix dactylifera Linn did not affect H.
pylori viability, authors such as Castillo et al., 2013, reported anti-Helicobacter
pylori activity from aqueous extracts of Persea americana Mill with
a minimum inhibitory concentration (MIC) > 1000 µg/mL tested with the
agar dilution method.
Methanolic extracts of plants used in this work have previously been tested
against H. pylori. The MIC of Ibervillea sonorae Green against
H. pylori is reported of 200 to 400 µg/mL (Robles-Zepeda et al.,
2011). Also, methanol extract of Persea americana Mill has been reported
as active with a MIC <7.5 µg/mL tested with the broth dilution method
(Castillo et al., 2013).
Asteraceae plants have been reported to contain essential oils, flavonoids (Wollenweber
et al., 1981), among other substances, and possess antimicrobial and antitumor
activities.
Our Asteraceae plant methanolic extracts showed a growth inhibition of H.
pylori ATCC 43504 at a concentration of 500 µg/mL, and the activity
was in a concentration-dependent manner. However the results obtained in this
research can not be compared with others due to the bioassay employed. The active
concentrations used in the present study can be considered high, nevertheless
there are active components which can inhibit the growth of the bacteria.
The MTT reduction assay was standardized in order to test the activity anti-Helicobacter
pylori of plants. It is possible that during the extraction method or when
the plants were boiled some trace amounts of compounds evaporated.
CONCLUSIONS
The methanolic extracts from a plant of the Asteraceae family showed up to 82.3%
H. pylori growth inhibition at a concentration of 500 µg/mL, which
warrants further studies on diverse in vitro and in vivo parameters.
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Autor
de correspondencia: David Espinosa Ramos.
alonsoespinosa81@hotmail.com
Artículo
recibido: 12 de Mayo de 2015.
Artículo aprobado para publicación: 26 de Junio de 2015.
Revista
Mexicana de Estomatología.
Vol 2, No
2 Enero - Junio 2015.
ISSN: 2007-9052
www.remexesto.com
ISSN: 2007-9052
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